THE AXIAL GRADIENTS IX HYDROZOA. 8l 



reducing parts soon show a retardation in staining in consequence 

 of decrease in concentration of permanganate in the region about 

 them. For these reasons the volume of solution used should be 

 large as compared with the volume of protoplasm and uniformity 

 of concentration at all points of the protoplasmic surfaces should 

 be maintained by continuous or frequent agitation. 



One difficulty exists in the technique of permanent preparation 

 of permanganate material either for whole mounts or for sections. 

 All clearing agents used thus far remove more or less rapidly the 

 black or brown deposit in the protoplasm, the MnO2 being 

 apparently soluble in or reacting in some way with the oils. 

 This disappearance of color also occurs in Canada balsam. In 

 the samples of clove oil used the disappearance of the color is 

 rapid, e.g., hydranths may fade from opaque black to light 

 uniform yellow in two or three days. In cedar oil the fading 

 occurs much more slowly and may be'a matter of weeks. In the 

 course of a study of permanganate preparations of larval stages 

 of echinoderms Mr. A. E. Galigher has found that with rapid 

 preparation sections could be obtained without appreciable loss 

 of color, but that in balsam the color gradually disappeared. 

 Small organisms, larval stages, etc., can be made sufficiently 

 translucent by dehydration and clearing so that the differences 

 in total amount of reduction are clearly visible and persist at 

 least for a day or two, or with some clearing agents much longer. 



It is scarcely necessary to point out that differences in thickness 

 of the layer of protoplasm through which the light is transmitted 

 may appear as differences in depth of staining. A hydroid stem 

 of larger diameter, for example, may appear to be more deeply 

 colored than a stem of smaller diameter and in. such cases it is 

 often quite impossible to determine whether the difference is real 

 or apparent. Such difficulties arise chiefly in connection with 

 preparations for total amount of reduction rather than with those 

 for rate of reduction, for in the latter reduction and coloration 

 occur first on the surface and progress inward, and the differences 

 in rate of staining can usually be seen in early stages on the 

 external surface of the protoplasm quite independently of its 

 thickness. 



